2-[(2-cyanoethyl)[4-[(6-nitrobenzothiazol-2-yl)azo]phenyl]amino]ethyl acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From November 16 to 29, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Source study has reliability 1. Details on the read across are attached in section 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:JN

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France Laboratories, Domaine des Oncins B.P.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at arrival: 18-19 grams
- Housing: up to 5 during acclimatisation; 1/cage during the study
- Diet: ad libitum throughout the study 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
- Water: ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: yes

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: approximately 15 to 20 air changes per hour
- Photoperiod: artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
- IN-LIFE DATES: from 16 to 29 November 2016

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Test item: 25, 10 and 5 % (w/w)
Positive control: 25 %

No. of animals per dose:

4 animals per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: a solubility trial was performed to evaluate the feasibility of test item formulations using acetone/olive oil 4:1 v/v, PEG 400 and DMSO as vehicle. A formulation suitable for application was obtained at a concentration of 25 % w/w in acetone/olive oil 4:1 v/v.
- Irritation: treated sites of all animals were examined daily (once before first dosing, before dosing on days 2 and 3 and daily thereafter).
- Systemic toxicity: animals were observed for clinical signs on day 1, before and approximately 1 hour after dosing; day 2 to 6, daily (approximately 1 hour after daily dosing, when applicable)
- Ear thickness measurements: ear thickness was measured by a suitable micrometer on day 1 (before dosing), on day 3 (before dosing) and on day 6.
- Erythema scores: irritation to the skin was assigned a numerical value according to the table below:
Erythema and eschar formation value
0 no erythema
1 very slight erythema
2 well defined erythema
3 moderate to severe erythema
4 severe erythema (beet redness) to eschar formation preventing grading of erythema

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LOCAL LYMPH NODE ASSAY (LLNA: BrdU-ELISA method)
- Criteria used to consider a positive response: test item is considered to induce sensitisation when the SI for any single treatment dose group is ≥ 1.6. It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: animals were treated for three consecutive days (days 1, 2, 3) with the vehicle, test or control item formulations. A dose volume of 25 µl/ear/day of each selected concentration and controls was applied to the dorsal surface of each ear (50 µl/animal/day), using a micropipette.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test.

Results and discussion

Positive control results:

In the group treated with the positive control item, a Stimulation Index of 2.56 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

3.03

Test group / Remarks:

low dose, 5.0 %

Results 2

Parameter:

SI

Value:

2.48

Test group / Remarks:

mid dose, 10 %

Results 3

Parameter:

SI

Value:

3.09

Test group / Remarks:

high dose, 25 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: a statistically significant increase in cell proliferation of draining lymph nodes was observed in all treatment groups.

DETAILS ON STIMULATION INDEX CALCULATION: the calculated Stimulation Indices (SI) were 3.03, 2.48 and 3.09, respectively at low, mid and high dose levels.

CLINICAL OBSERVATIONS: neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated of 25, 10 and 5 % (w/w).

BODY WEIGHTS: changes in body weight observed during the study were within the expected range for this strain and age of animals.

Applicant's summary and conclusion

Interpretation of results:

other: skin sensitiser in category 1 of the CLP Regulation (EC 1272/2008)

Conclusions:

A statistically significant increase in cell proliferation of draining lymph nodes was observed at all dose levels. The calculated Stimulation Indices (SI) were 3.03, 2.48 and 3.09, at the low, mid and high dose levels, i.e. 5, 10 and 25 %, respectively.
In the group treated with the positive control item, a Stimulation Index of 2.56 was calculated.

Executive summary:

Method

Test substance was assessed for the potential to cause skin sensitisation reactions following topical application to the skin of CBA/JN mice, using the LLNA:BrdU-ELISA method, according to the OECD guideline 442b.

In a preliminary test, concentrations of 25 (maximum feasible concentration), 10, 5, 2.5 and 1 % w/w in acetone: olive oil 4:1 (v/v) were tested to identify a non toxic and minimally irritant concentration and avoid false positive results.

No signs of toxicity (significant clinical signs or body weight losses) were observed at tested concentrations.

According to the results of the irritation screening, a concentration of 25 % w/w was judged to be not irritant.

In the main assay, test item was topically administered at concentrations of 25, 10 and 5 % (w/w), in acetone:olive oil 4:1 (v/v).

Results

No mortality nor clinical signs were recorded in any animal. Changes in bodyweight observed during the study were within the expected range for this strain and age of animals.

An increase in cell proliferation of draining lymph nodes was observed in the low, mid and high dose groups. This increase reached statistical significance in all dose levels (5, 10 and 25 % w/w). The calculated Stimulation Indices (SI) were 3.03, 2.48 and 3.09, at the low, mid and high dose levels (5, 10 and 25 %), respectively.

The above results indicate that test substance may elicit a sensitisation response in mice following dermal exposure, since in all dose groups the Stimulation Index was greater than 1.6.